Human nm23-like protein

ABSTRACT

The present invention provides polynucleotides which identify and encode a novel human nm23-like protein (H-nm23). The invention provides for genetically engineered expression vectors and host cells comprising the nucleic acid sequence encoding H-nm23 and for a method for producing the protein. The invention also provides for the use of substantially purified H-nm23 for the for the treatment of diseases associated with the expression of H-nm23. The invention also describes diagnostic assays which utilize diagnostic compositions comprising the polynucleotides which hybridize with naturally occurring sequences encoding H-nm23 and antibodies which specifically bind to the protein.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/460,532, filed Dec. 13, 1999, which is a divisionalapplication of U.S. application Ser. No. 09/199,842, filed Nov. 24,1998, now U.S. Pat. No. 6,087,125, issued Jul. 11, 2000, which is adivisional application of U.S. application Ser. No. 08/713,825, filedSep. 13, 1996, now U.S. Pat. No. 5,874,285, issued Feb. 23, 1999, all ofwhich applications and patents are hereby incorporated herein byreference.

FIELD OF THE INVENTION

[0002] The present invention relates to nucleic acid and amino acidsequences of a novel human nm23-like protein and to the use of thesesequences in the diagnosis, study, prevention and treatment of disease.

BACKGROUND OF THE INVENTION

[0003] The nm23 genes encode proteins that participate in thedevelopment and differentiation of normal tissues. Nm23 proteins arealso associated with the regulation of tumor metastasis. Homologs of thehighly conserved protein have been characterized in numerous tissuesincluding those from humans, mice, Drosophila, and Myxococcus xanthus.The nm23 proteins generally consist of 150 to 180 amino acid residues.All known nm23 proteins contain a leucine zipper motif and exhibitnucleoside diphosphate kinase (NDPK) activity.

[0004] Nm23 protein accumulation is coincident with the functionaldifferentiation of multiple epithelial tissues in the developing mouse.At the onset of organogenesis, the amount of nm23 protein is relativelylow and uniform throughout the mouse embryo. The protein begins toaccumulate preferentially in the first embryonic tissues todifferentiate, the developing nervous system and heart. Subsequentdifferentiation of liver, kidney, skin, intestine, adrenal, and stomachepithelial cells is accompanied by increased nm23 protein expression(Lakso M et al (1992) Cell Growth Differ 3:873-879).

[0005] In rodent, reduced expression of the nm23 gene systems has beencorrelated with increased potential for tumor metastasis. A suppressiveeffect of nm23 on several aspects of the cancer process, includingmetastasis, has been demonstrated in murine melanoma cells (Leone A etal (1991) Cell 65: 25-35). The number of metastases developed in amurine melanoma subline was inversely correlated with the expression oftwo murine isotypes, nm23-M1 and nm23-M2 (Baba H et al (1995) Cancer Res55:1977-1981).

[0006] In Drosophila, an nm23 homologue, abnormal wing discs (awd), isessential for normal development. Mutation or reduced expression of awdcauses abnormal tissue morphology, necrosis and widespread aberrantdifferentiation similar to malignant progression (Rosengard A M et al(1989) Nature 342:177-180).

[0007] Two human nm23 isoforms, nm23-H1 and nm23-H2, each consist of 152amino acid residues with Mr of 17,143 and 17,294, respectively. Theisoforms have 88% sequence identity and encode polypeptides identical tothe A and B chains of human erythrocyte NDPK (Gilles et al (1991) J BiolChem 266:8784-8789). NDPK is a hexameric enzyme, with isozymesconsisting of all combinations of the A and B chains (A6, A5B . . . AB5,B6). NDPK transfers a phosphoryl group between nucleoside tri- anddiphosphates via a covalent phosphoenzyme intermediate. In nm23-H1 and-H2, histidine 118 is the site of this transient phosphorylation.

[0008] Human nm23-H2 protein is also identical to the c-mycpurine-binding transcription factor PuF (Postel E H et al (1993) Science261:478-480). Myc, the protein product of the c-myc proto-oncogene, isproposed to modulate the expression of genes involved in cellularproliferation, differentiation, and tumor formation. Native PuF andpurified recombinant nm23-H2 bind to DNA sequences corresponding to anuclease-hypersensitive element (NHE) in the human c-myc P1 promoter,and induce accurate c-myc transcription in vitro.

[0009] The relationship between the DNA binding, transcriptionalactivation, and NDPK activities of nm23 was assessed by site-directedmutagenesis of recombinant nm23-H2. Although the NDPK phosphoenzymeactive site mutant H118F was inactive in NDPK assays, it displayednormal DNA binding affinity for the c-myc promoter and retained fullc-myc transcriptional activity in vitro (Postel E H and Ferrone C A(1994) J Biol Chem 269:8627-8630). This suggests that the DNAbinding/transcriptional activation and the NDPK activities of nm23-H2are independent properties of the nm23 proteins which may be associatedwith different biological functions.

[0010] The mechanism by which nm23 affect metastasis and development isunclear. Autophosphorylation of serine has been observed in nm23,distinct from NDPK-associated histidine phosphorylation (MacDonald N Jet al (1993) J Biol Chem 268:25780-25789). A direct correlation has beenobserved in mice between in vivo nm23 serine phosphorylation levels andsuppression of tumor metastatic potential among control and nm23-M1transfected murine melanoma cells. The serine phosphorylation of mousenm23 is inhibited by cAMP in vitro and forskolin in vivo, suggestingthat this phosphorylation is regulated by a signal transduction pathway.No correlation was found between nm23 NDPK activity and melanoma cellmetastasis, nor was NDPK activity inhibited by cAMP (MacDonald, supra).

[0011] Co-regulated expression of nm23 and mts1 (cyclin-dependentprotein kinase), another tumor-suppressor gene, alters the state oftubulin polymerization in B16 melanoma cell lines (Lakshmi M S et al(1993) Anticancer Res 13:299-303). The altered tubulin polymerization issuggested to impart invasive and metastasizing properties to the cellline.

[0012] Recently, a new human nm23 isoform, DR-nm23, has been cloned froma chronic myelogenous leukemia (CML) blast crisis cell line (VenturelliD et al (1995) Proc Natl Acad Sci USA 92:7435-7439). The DR-nm23 proteinconsists of 168 amino acids, and has 67% and 69% sequence identity tothe nm23-H1 and H2 isoforms, respectively. DR-nm23 is involved in normalhematopoiesis and, when overexpressed, may contribute to differentiationarrest, a feature of blastic transformation in CML.

[0013] The NM23 Gene Family and Cancer

[0014] Expression levels of nm23 have been monitored throughout thedevelopment and metastasis of several types of cancer. In some tumorstypes, an inverse correlation exists between expression of nm23 andmetastasis. For instance, elevated nm23 expression in human breastcancer tumors is associated with a decrease in lymph node metastasis andwith longer patient survival. The nm23 gene product may play animportant role in suppressing the metastatic phenotype (Hennessy et al(1991) J Natl Cancer Inst 83: 281-285). Stahl et al ((1991) Cancer Res51:445-449) report that metastatic breast tumors exhibit significantlyreduced levels of the nm23-H1 protein relative to the nm23-H2 protein.Tokunaga Y et al ((1993) Int J Cancer 55:66-71) likewise report thatexpression of nm23-H1, but not nm23-H2, is inversely associated withlymph-node metastasis. Overall survival is better in patients in whichnm23-H1 expression is elevated than in those in which it is lowered.Nm23-H1 therefore has value for predicting long-term survival of humanbreast-cancer patients.

[0015] Steeg P S et al ((1993) Breast Cancer Res Treat 25:175-187)report a significant association between reduced nm23 expression, at theRNA or protein levels, and aggressive tumor growth in human breastcancer. Decreases in nm23 expression begin prior to actualhistologically identifiable invasion. Expression of human nm23-H1 cDNAin the transfected metastatic human MDA-MB-435 breast carcinoma cellline suppresses metastatic potential by 50-90%. Transfection of humanbreast carcinoma cell lines with a nm23-H1 expression vector restoresmany phenotypically normal features to these cells (Howlett A R et al(1994) J Natl Cancer Inst 86: 1838-1844).

[0016] Differences in expression levels of nm23-H1 have been reportedamong normal ovary tissue, benign tumors and carcinomas. The nm23-H1protein is absent from metastatic ovarian carcinomas more often thannon-metastatic carcinomas (Kapitanovic S et al (1995) Anticancer Res15:587-590). Tumors that do not metastasize into the lymph nodes expressnm23-H1 at significantly higher levels than tumors that do metastasizeinto the lymph nodes. These observations suggest that the nm23-H1protein may have an inhibitory effect on lymphatic metastasis (Viel A etal (1995) Cancer Res 55:2645-2650).

[0017] Low levels of nm23 mRNA also correlate with high metastaticpotential in malignant melanomas (Florenes V A et al (1992) Cancer Res52: 6088-91). However, patients with higher nm23 expression inmetastatic tissue tend to live longer (Xerri L et al (1994) Br J Cancer70:1224-1228).

[0018] Similar correlations between nm23 and metastasis are reported forhepatocellular carcinomas (Iizuka et al. 95), prostate cancer (Fishmanet al. 94), and leukemia cell lines (Yamashiro et al. 94).

[0019] However, among other tumor types, nm23 expression has no apparentrelationship to metastatic potential. Expression of nm23 even correlatesdirectly with severity in some of these cancers. For instance, nm23expression in various types of thyroid cancers does not appear to play arole in metastasis. The average level of nm23 gene expression in stagesI through III of differentiated thyroid carcinoma is comparable to thatin multinodular goiters. In advanced stages of thyroid carcinoma (stageIV and anaplastic), a 2-fold increase in nm23 expression is observed.This suggests a direct correlation of nm23 expression with rapid cellproliferation in thyroid cancer (Zou M et al (1993) Br J Cancer68:385-388).

[0020] Yamaguchi A et al ((1993) Br J Cancer 68:1020-1024) report thatno significant correlation exists between nm23-H1 expression andcolorectal tumor histology, serosal invasion, lymphatic invasion, venousinvasion, or lymph node metastasis. However, Zeng Z S et al ((1994) Br JCancer 70:1025-1030) report that nm23-H1 expression increases with localcolorectal tumor severity, and in liver metastases even higher levels ofnm23-H1 expression are found. In addition, Zeng et al (supra) observedtwo immunoreactive species of nm23-H1 expressed in roughly equalproportions in 16 patients: the predicted 17 kDa form and a larger 18.5kDa form.

[0021] High expression levels of a 19 kDa form of nm23-H1 protein inneuroblastoma are associated with advanced stage (III and IV) diseaseand with N-myc gene amplification (Hailat N et al (1991) J Clin Invest88: 341-345).

[0022] Engel M et al ((1993) Int J Cancer 55:375-9) show that highlevels of nm23-H1 and nm23-H2 mRNA in human squamous-cell lungcarcinoma, large-cell carcinoma, sarcoma, and carcinoids are associatedwith poor differentiation, advanced stage tumors and poor prognosis. Inhuman renal carcinoma cell lines and in high stage renal cancers, levelsof nm23-H1 and nm23-H2 mRNAs were elevated (Kanayama H et al (1994) IntJ Urol 1: 324-331).

[0023] The discovery of DR-nm23 (Venturelli D et al, supra) raises thepossibility that additional nm23-like proteins present in human tissuesmediate normal or abnormal cellular processes within those tissues.Along with its diagnostic and therapeutic value, the nm23 proteins havea pronounced prognostic value for those types of tumors in which arelationship between expression of nm23 and metastasis exist. Nm23 isalso useful in diagnostic, prognostic and therapeutic applications forthose tumor types for which levels of expression correlate with tumorseverity. Therefore, the selective modulation of the expression oractivity of a novel nm23-like protein may allow the successful treatmentof amenable types of cancer.

SUMMARY OF THE INVENTION

[0024] The present invention discloses a novel nm23-like protein,hereinafter referred to as H-nm23, having chemical and structuralhomology to human nm23 isoforms DR-nm23, nm23-H1 and nm23-H2.Accordingly, the invention features a substantially purified H-nm23,encoded by amino acid sequence of SEQ ID NO:1, having structuralcharacteristics of the family of nm23 proteins.

[0025] One aspect of the invention features isolated and substantiallypurified polynucleotides which encode H-nm23. In a particular aspect,the polynucleotide is the nucleotide sequence of SEQ ID NO:2. Inaddition, the invention features nucleotide sequences which hybridizeunder stringent conditions to SEQ ID NO:2.

[0026] The invention further relates to nucleic acid sequence encodingH-nm23, oligonucleotides, peptide nucleic acids (PNA), fragments,portions or antisense molecules thereof. The present invention alsorelates to an expression vector which includes polynucleotide encodingH-nm23 and its use to transform host cells or organisms. The inventionalso relates to antibodies which bind specifically to the nm23 havingamino acid sequence of SEQ ID NO:1 and to a pharmaceutical compositioncomprising a substantially purified nm23 having amino acid sequence ofSEQ ID NO:1.

BRIEF DESCRIPTION OF THE FIGURES

[0027]FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1)and the nucleic acid sequence (SEQ ID NO:2) of the human nm23-likeprotein H-nm23. The alignment was produced using MACDNASIS software(Hitachi Software Engineering Co Ltd, San Bruno, Calif.).

[0028]FIGS. 2A and 2B show the amino acid sequence alignments amongH-nm23 (SEQ ID NO:1), the human nm23 isoforms DR-nm23 (GI 1051256; SEQID NO:3), nm23-H1 (GI 468542; SEQ ID NO:4), and nm23-H2 (GI 127983, SEQID NO:5). The multisequence alignment program of DNASTAR software(DNAStar Inc, Madison, Wis.) was used.

[0029]FIG. 3 shows the hydrophobicity plot (generated using MACDNASISsoftware) for H-nm23, SEQ ID NO:1; the X axis reflects amino acidposition, and the negative Y axis, hydrophobicity.

[0030]FIGS. 4A and 4B are a list of Incyte libraries in whichfull-length or partial H-nm23 mRNA sequences are represented, theabundance of the sequences, and the percent abundance.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Definitions

[0032] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represent the sense or antisense strand.Similarly, amino acid sequence as used herein refers to peptide orprotein sequence.

[0033] “Consensus” as used herein may refer to a nucleic acidsequence 1) which has been resequenced to resolve uncalled bases, 2)which has been extended using XL-PCR (Perkin Elmer) in the 5′ or the 3′direction and resequenced, 3) which has been assembled from theoverlapping sequences of more than one Incyte clone GCG FragmentAssembly System, (GCG, Madison, Wis.), or 4) which has been bothextended and assembledPeptide nucleic acid” as used herein refers to amolecule which comprises an oligomer to which an amino acid residue,such as lysine, and an amino group have been added. These smallmolecules, also designated anti-gene agents, stop transcript elongationby binding to their complementary (template) strand of nucleic acid(Nielsen P E et al (1993) Anticancer Drug Des 8:53-63).

[0034] A “variant” of H-nm23 is defined as an amino acid sequence thatis different by one or more amino acid substitutions. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software.

[0035] A “deletion” is defined as a change in either nucleotide or aminoacid sequence in which one or more nucleotides or amino acid residues,respectively, are absent.

[0036] An “insertion” or “addition” is that change in a nucleotide oramino acid sequence which has resulted in the addition of one or morenucleotides or amino acid residues, respectively, as compared to thenaturally occurring H-nm23.

[0037] A “substitution” results from the replacement of one or morenucleotides or amino acids by different nucleotides or amino acids,respectively.

[0038] The term “biologically active” refers to a H-nm23 havingstructural, regulatory or biochemical functions of the naturallyoccurring H-nm23. Likewise, “immunologically active” defines thecapability of the natural, recombinant or synthetic H-nm23, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0039] The term “derivative” as used herein refers to the chemicalmodification of a nucleic acid encoding H-nm23 or the encoded H-nm23.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics ofnatural H-nm23.

[0040] As used herein, the term “substantially purified” refers tomolecules, either nucleic or amino acid sequences, that are removed fromtheir natural environment, isolated or separated, and are at least 60%free, preferably 75% free, and most preferably 90% free from othercomponents with which they are naturally associated.

[0041] “Stringency” typically occurs in a range from about Tm-5° C. (5°C. below the Tm of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, a stringencyhybridization can be used to identify or detect identical polynucleotidesequences or to identify or detect similar or related polynucleotidesequences.

[0042] The term “hybridization” as used herein shall include “anyprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing” (Coombs J (1994) Dictionary ofBiotechnology, Stockton Press, New York, N.Y.). Amplification as carriedout in the polymerase chain reaction technologies is described inDieffenbach C W and G S Dveksler (1995, PCR Primer, a Laboratory Manual,Cold Spring Harbor Press, Plainview, N.Y.).

[0043] Preferred Embodiment

[0044] The present invention relates to a novel human nm23-like protein,designated H-nm23, initially identified among the partial cDNAs from ahuman breast tissue library (BRSTNOT05) and to the use of the nucleicacid and amino acid sequences disclosed herein in the study, diagnosis,prevention and treatment of disease. Northern analysis using theLIFESEQ™ database (Incyte Pharmaceuticals, Palo Alto, Calif.) shows thatmRNA encoding H-nm23 was found in tissues isolated from small intestine,prostate, ovary, lymph node, breast spinal cord, thyroid, colon, lungand bladder tissues (FIGS. 4A and 4B). Of the 41 cDNA librariescontaining H-nm23 mRNA, ten were derived from prostate tissue. Five ofthese libraries were from prostate tumors. Of the five non-tumorprostate libraries, four were generated from non-cancerous tissuesexcised from cancer patients, and the remaining library was derived froma young man asymptomatic of cancer. H-nm23 mRNA was also found in fetaland neonatal heart, spleen, lung, and kidney, but was not found in theseorgans in adults, indicating that H-nm23 may be related to thedevelopment of these tissues. The single exception to this trend was thepresence of H-nm23 in adult heart myoma (atrial myxoma), a benign tumor.H-nm23 mRNA was also transcribed in the hNT2 cell line, which wasderived from a human teratocarcinoma that exhibited propertiescharacteristic of a committed neuronal precursor at an early stage ofdevelopment.

[0045] The present invention also encompasses H-nm23 variants. Apreferred H-nm23 variant is one having at least 80% amino acid sequencesimilarity to the H-nm23 amino acid sequence (SEQ ID NO:1), a morepreferred H-nm23 variant is one having at least 90% amino acid sequencesimilarity to SEQ ID NO:1 and a most preferred H-nm23 variant is onehaving at least 95% amino acid sequence similarity to SEQ ID NO:1.

[0046] The nucleic acid sequence encoding a portion of H-nm23 was firstidentified in the cDNA, Incyte Clone 964996, through acomputer-generated search for amino acid sequence alignments. Thenucleic acid sequence, SEQ ID NO:2, disclosed herein (FIGS. 1A, 1B, and1C) encodes the amino acid sequence, SEQ ID NO:1, designated H-nm23. Thefull length cDNA was assembled from Incyte Clones 603550 (BRSTTUT01);668015 (SCORNOT01); 669792 (CRBLNOT01); 964996 (BRSTNOT05); 1211471(BRSTNOT02); 1312720 (BLADTUT02); and 1403767 (LATRTUT02) from theLIFESEQ™ database (Incyte Pharmaceuticals, Palo Alto, Calif.).

[0047] The present invention is based in part on the structural homologyshown in FIGS. 2A and 2B, among H-nm23 and other human nm23 isoformsincluding DR-nm23 (GI 1051256; Venturelli D et al, supra), nm23-H1 (GI468542, Rosengard A M et al, supra) and nm23-H2 (GI 127983, Stahl J A etal, supra). DR-nm23, nm23-H1, and nm23-H2 have, respectively, 50%, 46%and 44% amino acid sequence identity to H-nm23.

[0048] The H-nm23 protein sequence consists of 187 amino acids. Frompositions 68 to 97 the amino acid sequence contains a periodicrepetition of leucine residues at every sixth to seventh position. Thispattern is characteristic of a leucine zipper motif, which is present inmany gene regulatory proteins (Busch S J et al (1990) Trends Genet6:36-40). H-nm23 has an arg-gly-asp sequence at positions 138-140. Thissequence, known as the “RGD motif”, is crucial in the interaction offibronectin with its integrin cell-surface receptor, and plays a role incell adhesion (d'Souza S E et al (1991) Trends Biochem Sci 16:246-250).H-nm23 also contains the consensus sequence for NDPK (Gilles A M et al,supra) which includes the active site histidine at position 151.

[0049] The H-nm23 Coding Sequences

[0050] The nucleic acid and amino acid sequences of H-nm23 are shown inFIGS. 1A, 1B, and 1C. In accordance with the invention, any nucleic acidsequence which encodes the amino acid sequence of H-nm23 can be used togenerate recombinant molecules which express H-nm23. In a specificembodiment described herein, a partial sequence of H-nm23 was firstisolated as Incyte Clone 964996 from a human breast tissue library(BRSTNOT05).

[0051] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding H-nm23, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. The invention contemplates each and every possible variationof nucleotide sequence that could be made by selecting combinationsbased on possible codon choices. These combinations are made inaccordance with the standard triplet genetic code as applied to thenucleotide sequence of naturally occurring H-nm23, and all suchvariations are to be considered as being specifically disclosed.

[0052] Although nucleotide sequences which encode H-nm23 and itsvariants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring H-nm23 under appropriately selectedconditions of stringency, it may be advantageous to produce nucleotidesequences encoding H-nm23 or its derivatives possessing a substantiallydifferent codon usage. Codons may be selected to increase the rate atwhich expression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Other reasons forsubstantially altering the nucleotide sequence encoding H-nm23 and itsderivatives without altering the encoded amino acid sequences includethe production of RNA transcripts having more desirable properties, suchas a greater half-life, than transcripts produced from the naturallyoccurring sequence.

[0053] It is now possible to produce a DNA sequence, or portionsthereof, encoding a H-nm23 and its derivatives entirely by syntheticchemistry, after which the synthetic gene may be inserted into any ofthe many available DNA vectors and cell systems using reagents that arewell known in the art at the time of the filing of this application.Moreover, synthetic chemistry may be used to introduce mutations into agene encoding H-nm23.

[0054] Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequence of FIGS. 1A, 1B, and 1C under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Berger and Kimmel (1987, Guide to Molecular Cloning Techniques,Methods in Enzymology, Vol 152, Academic Press, San Diego, Calif.)incorporated herein by reference, and confer may be used at a definedstringency.

[0055] Altered nucleic acid sequences encoding H-nm23 which may be usedin accordance with the invention include deletions, insertions orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent H-nm23. The proteinmay also show deletions, insertions or substitutions of amino acidresidues which produce a silent change and result in a functionallyequivalent H-nm23. Deliberate amino acid substitutions may be made onthe basis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of H-nm23 is retained. For example, negativelycharged amino acids include aspartic acid and glutamic acid; positivelycharged amino acids include lysine and arginine; and amino acids withuncharged polar head groups having similar hydrophilicity values includeleucine, isoleucine, valine; glycine, alanine; asparagine, glutamine;serine, threonine phenylalanine, and tyrosine.

[0056] Included within the scope of the present invention are alleles ofH-nm23. As used herein, an “allele” or “allelic sequence” is analternative form of H-nm23. Alleles result from a mutation, i.e., achange in the nucleic acid sequence, and generally produce altered mRNAsor polypeptides whose structure or function may or may not be altered.Any given gene may have none, one or many allelic forms. Commonmutational changes which give rise to alleles are generally ascribed tonatural deletions, additions or substitutions of amino acids. Each ofthese types of changes may occur alone, or in combination with theothers, one or more times in a given sequence.

[0057] Methods for DNA sequencing are well known in the art and employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (USBiochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer,Norwalk, Conn.), thermostable T7 polymerase (Amersham, Chicago, Ill.),or combinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg, Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), PeltierThermal Cycler (PTC200; M J Research, Watertown, Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

[0058] Extending the Polynucleotide Sequence

[0059] The polynucleotide sequence encoding H-nm23 may be extendedutilizing partial nucleotide sequence and various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. Gobinda et al (1993; PCR Methods Applic 2:318-22) disclose“restriction-site” polymerase chain reaction (PCR) as a direct methodwhich uses universal primers to retrieve unknown sequence adjacent to aknown locus. First, genonic DNA is amplified in the presence of primerto a linker sequence and a primer specific to the known region. Theamplified sequences are subjected to a second round of PCR with the samelinker primer and another specific primer internal to the first one.Products of each round of PCR are transcribed with an appropriate RNApolymerase and sequenced using reverse transcriptase.

[0060] Inverse PCR can be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia T et al (1988)Nucleic Acids Res 16:8186). The primers may be designed using OLIGO®4.06 Primer Analysis Software (1992; National Biosciences Inc, Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0061] Capture PCR (Lagerstrom M et al (1991) PCR Methods Applic1:111-19) is a method for PCR amplification of DNA fragments adjacent toa known sequence in human and yeast artificial chromosome DNA. CapturePCR also requires multiple restriction enzyme digestions and ligationsto place an engineered double-stranded sequence into an unknown portionof the DNA molecule before PCR.

[0062] Another method which may be used to retrieve unknown sequences isthat of Parker J D et al (1991; Nucleic Acids Res 19:3055-60).Additionally, one can use PCR, nested primers and PROMOTERFINDERlibraries to walk in genonic DNA (Clontech (Palo Alto, Calif.)). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0063] Preferred libraries for screening for full length cDNAs are onesthat have been size-selected to include larger cDNAs. Also, randomprimed libraries are preferred in that they will contain more sequenceswhich contain the 5′ and upstream regions of genes. A randomly primedlibrary may be particularly useful if an oligo d(T) library does notyield a full-length CDNA. Genomic libraries are useful for extensioninto the 5′ nontranslated regulatory region.

[0064] Capillary electrophoresis may be used to analyze the size orconfirm the nucleotide sequence of sequencing or PCR products. Systemsfor rapid sequencing are available from Perkin Elmer, BeckmanInstruments (Fullerton, Calif.), and other companies. Capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity is converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR from Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display is computercontrolled. Capillary electrophoresis is particularly suited to thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample. The reproducible sequencing of up to 350bp of M13 phage DNA in 30 min has been reported (Ruiz-Martinez M C et al(1993) Anal Chem 65:2851-8).

[0065] Expression of the Nucleotide Sequence

[0066] In accordance with the present invention, polynucleotidesequences which encode H-nm23, fragments of the polypeptide, fusionproteins or functional equivalents thereof may be used in recombinantDNA molecules that direct the expression of H-nm23 in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences which encode substantially the same or a functionallyequivalent amino acid sequence, may be used to clone and express H-nm23.As will be understood by those of skill in the art, it may beadvantageous to produce H-nm23-encoding nucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particularprokaryotic or eukaryotic host (Murray E et al (1989) Nuc Acids Res17:477-508) can be selected, for example, to increase the rate of H-nm23expression or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, than transcripts produced fromnaturally occurring sequence.

[0067] The nucleotide sequences of the present invention can beengineered in order to alter a coding sequence of H-nm23 for a varietyof reasons, including but not limited to, alterations which modify thecloning, processing and/or expression of the gene product. For example,mutations may be introduced using techniques which are well known in theart, e.g., site-directed mutagenesis to insert new restriction sites, toalter glycosylation patterns, to change codon preference, to producesplice variants, etc.

[0068] In another embodiment of the invention, a natural, modified orrecombinant nucleotide sequence encoding H-nm23 may be ligated to aheterologous sequence to encode a fusion protein. For example, forscreening of peptide libraries for inhibitors of H-nm23 activity, it maybe useful to encode a chimeric H-nm23 protein that is recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between a H-nm23 sequence and theheterologous protein sequence, so that the H-nm23 may be cleaved andsubstantially purified away from the heterologous moiety.

[0069] In an alternate embodiment of the invention, the coding sequencefor H-nm23 may be synthesized, whole or in part, using chemical methodswell known in the art (see Caruthers M H et al (1980) Nuc Acids Res SympSer 215-23, Horn T et al(1980) Nuc Acids Res Symp Ser 225-32, etc).Alternatively, the protein itself could be produced using chemicalmethods to synthesize a H-nm23 amino acid sequence, whole or in part.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge J Y et al (1995) Science 269:202-204)and automated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer) in accordance with the instructionsprovided by the manufacturer.

[0070] The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo, New York N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally the amino acidsequence of H-nm23, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0071] Expression Systems

[0072] In order to express a biologically active H-nm23, the nucleotidesequence encoding H-nm23 or its functional equivalent, is inserted intoan appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0073] Methods which are well known to those skilled in the art can beused to construct expression vectors containing a H-nm23 coding sequenceand appropriate transcriptional or translational controls. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination or genetic recombination. Such techniques aredescribed in Sambrook et al (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y. and Ausubel F M et al(1989) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y.

[0074] A variety of expression vector/host systems may be utilized tocontain and express a H-nm23 coding sequence. These include but are notlimited to microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransfected with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterialexpression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.

[0075] The “control elements” or “regulatory sequences” of these systemsvary in their strength and specificities and are those nontranslatedregions of the vector, enhancers, promoters, and 3′ untranslatedregions, which interact with host cellular proteins to carry outtranscription and translation. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 (Gibco BRL) and ptrp-lac hybrids and the likemay be used. The baculovirus polyhedrin promoter may be used in insectcells. Promoters or enhancers derived from the genomes of plant cells(e.g., heat shock, RUBISCO; and storage protein genes) or from plantviruses (e.g., viral promoters or leader sequences) may be cloned intothe vector. In mammalian cell systems, promoters from the mammaliangenes or from mammalian viruses are most appropriate. If it is necessaryto generate a cell line that contains multiple copies of H-nm23, vectorsbased on SV40 or EBV may be used with an appropriate selectable marker.

[0076] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for H-nm23. For example, whenlarge quantities of H-nm23 are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be desirable. Such vectors include, but are notlimited to, the multifunctional E. coli cloning and expression vectorssuch as BLUESCRIPT (Stratagene), in which the H-nm23 coding sequence maybe ligated into the vector in frame with sequences for theamino-terminal Met and the subsequent 7 residues of β-galactosidase sothat a hybrid protein is produced; pIN vectors (Van Heeke & Schuster(1989) J Biol Chem 264:5503-5509); and the like. pGEX vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems are designedto include heparin, thrombin or factor XA protease cleavage sites sothat the cloned polypeptide of interest can be released from the GSTmoiety at will.

[0077] In the yeast, Saccbaromyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase and PGH may be used. For reviews, see Ausubel et al(supra) and Grant et al (1987) Methods in Enzymology 153:516-544.

[0078] In cases where plant expression vectors are used, the expressionof a sequence encoding H-nm23 may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV (Brisson et al (1984) Nature 310:511-514) may be usedalone or in combination with the omega leader sequence from TMV(Takamatsu et al (1987) EMBO J 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO (Coruzzi et al (1984)EMBO J 3:1671-1680; Broglie et al (1984) Science 224:838-843); or heatshock promoters (Winter J and Sinibaldi R M (1991) Results Probl CellDiffer 17:85-105) may be used. These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. For reviews of such techniques, see Hobbs S or Murry L Ein McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill NewYork, N.Y., pp 191-196 or Weissbach and Weissbach (1988) Methods forPlant Molecular Biology, Academic Press, New York, N.Y., pp 421-463.

[0079] An alternative expression system which could be used to expressH-nm23 is an insect system. In one such system, Autographa californicanuclear polyhedrosis virus (AcNPV) is used as a vector to expressforeign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.The H-nm23 coding sequence may be cloned into a nonessential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the H-nm23 coding sequencewill render the polyhedrin gene inactive and produce recombinant viruslacking coat protein coat. The recombinant viruses are then used toinfect S. frugiperda cells or Trichoplusia larvae in which H-nm23 isexpressed (Smith et al (1983) J Virol 46:584; Engelhard E K et al (1994)Proc Nat Acad Sci 91:3224-7).

[0080] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, a coding sequence for H-nm23 may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a nonessential E1or E3 region of the viral genome will result in a viable virus capableof expressing H-nm23 in infected host cells (Logan and Shenk (1984) ProcNatl Acad Sci 81:3655-59). In addition, transcription enhancers, such asthe rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mainalian host cells.

[0081] Specific initiation signals may also be required for efficienttranslation of a H-nm23 sequence. These signals include the ATGinitiation codon and adjacent sequences. In cases where nucleic acidencoding H-nm23, its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly coding sequence, or a portion thereof, is inserted, exogenoustranscriptional control signals including the ATG initiation codon mustbe provided. Furthermore, the initiation codon must be in the correctreading frame to ensure transcription of the entire insert. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate to the cell system inuse (Scharf D et al (1994) Results Probl Cell Differ 20:125-62; Bittneret al (1987) Methods in Enzymol 153:516-544).

[0082] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, etchave specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

[0083] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress H-nm23 may be transformed using expression vectors which containviral origins of replication or endogenous expression elements and aselectable marker gene. Following the introduction of the vector, cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clumps of stably transformed cells can be proliferated usingtissue culture techniques appropriate to the cell type.

[0084] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler M et al (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy I et al (1980)Cell 22:817-23) genes which can be employed in tk- or aprt- cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler M et al (1980) Proc Natl Acad Sci77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin F et al (1981) J Mol Biol 150:1-14)and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman SC and R C Mulligan (1988) Proc Natl Acad Sci 85:8047-51). Recently, theuse of visible markers has gained popularity with such markers asanthocyanins, β glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin, being widely used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes C Aet al (1995) Methods Mol Biol 55:121-131).

[0085] Identification of Transformants Containing the PolynucleotideSequence

[0086] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionshould be confirmed. For example, if the H-nm23 polynucleotide sequenceis inserted within a marker gene sequence, recombinant cells containingH-nm23 can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with a H-nm23sequence under the control of a single promoter. Expression of themarker gene in response to induction or selection usually indicatesexpression of the tandem H-nm23 as well.

[0087] Alternatively, host cells which contain the coding sequence forH-nm23 and express H-nm23 may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay orimmunoassay techniques which include membrane, solution, or chip basedtechnologies for the detection and/or quantification of the nucleic acidor protein.

[0088] The presence of the polynucleotide sequence encoding H-nm23 canbe detected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of H-nm23-encoding nucleotides. Nucleicacid amplification based assays involve the use of oligonucleotides oroligomers based on the H-nm23 sequence to detect transformantscontaining H-nm23 DNA or RNA. As used herein “oligonucleotides” or“oligomers” refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides which can beused as a probe or amplimer.

[0089] A variety of protocols for detecting and measuring the expressionof H-nm23, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson H-nm23 is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton Ret al (1990, Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox D E et al (1983, J Exp Med 158:1211).

[0090] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to H-nm23 include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the H-nm23 sequence, or any portion of it,may be cloned into a vector for the production of an mRNA probe. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3 or SP6 and labeled nucleotides.

[0091] A number of companies such as Pharmacia Biotech (Piscataway,N.J.), Promega (Madison, Wis.), and US Biochemical Corp (Cleveland,Ohio) supply commercial kits and protocols for these procedures.Suitable reporter molecules or labels include those radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241. Also, recombinant immunoglobulins may be produced as shown inU.S. Pat. No. 4,816,567 incorporated herein by reference.

[0092] Purification of H-nm23

[0093] Host cells transformed with a H-nm23-encoding nucleotide sequencemay be cultured under conditions suitable for the expression andrecovery of the encoded protein from cell culture. The protein producedby a recombinant cell may be contained intracellularly or secreteddepending on the sequence and/or the vector used. As will be understoodby those of skill in the art, expression vectors containing H-nm23 canbe designed for efficient production and proper transmembrane insertionof H-nm23 into a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may join H-nm23 to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins (Kroll D J et al (1993) DNA Cell Biol 12:441-53; cfdiscussion of vectors infra containing fusion proteins).

[0094] H-nm23 may also be expressed as a recombinant protein with one ormore additional polypeptide domains added to facilitate proteinpurification. Such purification facilitating domains include, but arenot limited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle, Wash.). The inclusion of a cleavable linker sequencessuch as Factor XA or enterokinase (Invitrogen, San Diego, Calif.)between the purification domain and H-nm23 is useful to facilitatepurification. One such expression vector provides for expression of afusion protein compromising an H-nm23 and contains nucleic acid encoding6 histidine residues followed by thioredoxin and an enterokinasecleavage site. The histidine residues facilitate purification on IMAC(immobilized metal ion affinity chromatography as described in Porath etal (1992) Protein Expression and Purification 3: 263-281) while theenterokinase cleavage site provides a means for purifying the H-nm23from the fusion protein.

[0095] In addition to recombinant production, fragments of H-nm23 may beproduced by direct peptide synthesis using solid-phase techniques (cfStewart et al (1969) Solid-Phase Peptide Synthesis, W H Freeman Co, SanFrancisco; Merrifield J (1963) J Am Chem Soc 85:2149-2154). In vitroprotein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City,Calif.) in accordance with the instructions provided by themanufacturer. Various fragments of H-nm23 may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

[0096] Uses of H-nm23

[0097] The rationale for the use of polynucleotide and polypeptidesequences disclosed herein is based in part on the chemical andstructural homology among the novel H-nm23 and human isoforms of nm23.H-nm23 may be used in the diagnosis, prognosis and treatment of diseasesassociated with abnormal tissue development and differentiationincluding cancer.

[0098] H-nm23 may be useful as a tumor suppressor or a metastaticinhibitor, decreasing the severity or the metastatic potential ofcertain tumor types.

[0099] A correlation between expression of H-nm23 and metastasis inspecific tumors would provide a measure of metastatic potential. In suchtumor types, H-nm23 may be useful as a prognostic marker. Antibodiesspecifically recognizing H-nm23 may be used to quantitate H-nm23 forprognostic and diagnostic purposes.

[0100] H-nm23 or its fragments can be used to identify specificmolecules with which it interacts. Such molecules include agonists thatenhance H-nm23 activity. Furthermore, DNA segments to which H-nm23 bindsmay reveal promoter regions or other regulatory sites important in thecontrol of cell cycle or tumor-associated genes.

[0101] H-nm23 Antibodies

[0102] H-nm23-specific antibodies are useful for the diagnosis andprognosis of conditions and diseases associated with expression ofH-nm23. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments and fragments producedby a Fab expression library. Neutralizing antibodies, i.e., those whichinhibit dimer formation, are especially preferred for diagnostics andtherapeutics.

[0103] H-nm23 for antibody induction does not require biologicalactivity; however, the protein fragment, or oligopeptide must beantigenic. Peptides used to induce specific antibodies may have an aminoacid sequence consisting of at least five amino acids, preferably atleast 10 amino acids. Preferably, they should mimic a portion of theamino acid sequence of the natural protein and may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of H-nm23 amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule. Procedures well known in the art can be used forthe production of antibodies to H-nm23.

[0104] For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc may be immunized by injection with H-nm23 orany portion, fragment or oligopeptide which retains immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvumare potentially useful human adjuvants.

[0105] Monoclonal antibodies to H-nm23 may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Koehler and Milstein(1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosboret al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985)Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New York,N.Y., pp 77-96).

[0106] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al (1984) Proc Natl AcadSci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda etal (1985) Nature 314:452-454). Alternatively, techniques described forthe production of single chain antibodies (U.S. Pat. No. 4,946,778) canbe adapted to produce H-nm23-specific single chain antibodies.

[0107] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G andMilstein C (1991; Nature 349:293-299).

[0108] Antibody fragments which contain specific binding sites forH-nm23 may also be generated. For example, such fragments include, butare not limited to, the F(ab′)2 fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)2fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse W D et al (1989) Science 256:1275-1281).

[0109] A variety of protocols for competitive binding orimmunoradiometric assays using either polyclonal or monoclonalantibodies with established specificities are well known in the art.Such immunoassays typically involve the formation of complexes betweenH-nm23 and its specific antibody and the measurement of complexformation. A two-site, monoclonal-based immunoassay utilizing monoclonalantibodies reactive to two noninterfering epitopes on a specific H-nm23protein is preferred, but a competitive binding assay may also beemployed. These assays are described in Maddox D E et al (1983, J ExpMed 158:1211).

[0110] Diagnostic Assays Using H-nm23 Specific Antibodies

[0111] Particular H-nm23 antibodies are useful for the diagnosis ofconditions or diseases characterized by expression of H-mn3 or in assaysto monitor patients being treated with H-nm23, agonists or inhibitors.Diagnostic assays for H-nm23 include methods utilizing the antibody anda label to detect H-nm23 in human body fluids or extracts of cells ortissues. The polypeptides and antibodies of the present invention may beused with or without modification. Frequently, the polypeptides andantibodies will be labeled by joining them, either covalently ornoncovalently, with a reporter molecule. A wide variety of reportermolecules are known, several of which were described above.

[0112] A variety of protocols for measuring H-nm23, using eitherpolyclonal or monoclonal antibodies specific for the respective proteinare known in the art. Examples include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting(FACS). A two-site, monoclonal-based immunoassay utilizing monoclonalantibodies reactive to two non-interfering epitopes on H-nm23 ispreferred, but a competitive binding assay may be employed. These assaysare described, among other places, in Maddox, D E et al (1983, J Exp Med158:1211).

[0113] In order to provide a basis for diagnosis, normal or standardvalues for H-nm23 expression must be established. This is accomplishedby combining body fluids or cell extracts taken from normal subjects,either animal or human, with antibody to H-nm23 under conditionssuitable for complex formation which are well known in the art. Theamount of standard complex formation may be quantified by comparingvarious artificial membranes containing known quantities of H-nm23 withboth control and disease samples from biopsied tissues. Then, standardvalues obtained from normal samples may be compared with values obtainedfrom samples from subjects potentially affected by disease. Deviationbetween standard and subject values establishes the presence of diseasestate.

[0114] Drug Screening

[0115] H-nm23, its catalytic or immunogenic fragments or oligopeptidesthereof, can be used for screening therapeutic compounds in any of avariety of drug screening techniques. The fragment employed in such atest may be free in solution, affixed to a solid support, borne on acell surface, or located intracellularly. The formation of bindingcomplexes, between H-nm23 and the agent being tested, may be measured.

[0116] Another technique for drug screening which may be used for highthroughput screening of compounds having suitable binding affinity tothe H-nm23 is described in detail in “Determination of Amino AcidSequence Antigenicity” by Geysen H M, WO Application 84/03564, publishedon Sep. 13, 1984, and incorporated herein by reference. In summary,large numbers of different small peptide test compounds are synthesizedon a solid substrate, such as plastic pins or some other surface. Thepeptide test compounds are reacted with fragments of H-nm23 and washed.Bound H-nm23 is then detected by methods well known in the art.Substantially purified H-nm23 can also be coated directly onto platesfor use in the aforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

[0117] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingH-nm23 specifically compete with a test compound for binding H-nm23. Inthis manner, the antibodies can be used to detect the presence of anypeptide which shares one or more antigenic determinants with H-nm23.

[0118] Uses of the Polynucleotide Encoding H-nm23

[0119] A polynucleotide encoding H-nm23, or any part thereof, may beused for diagnostic and/or therapeutic purposes. For diagnosticpurposes, the H-nm23 of this invention may be used to detect andquantitate gene expression in biopsied tissues in which expression ofH-nm23 may be implicated. The diagnostic assay is useful to distinguishbetween absence, presence, and excess expression of H-nm23 and tomonitor regulation of H-nm23 levels during therapeutic intervention.Included in the scope of the invention are oligonucleotide sequences,antisense RNA and DNA molecules, and PNAs.

[0120] Another aspect of the subject invention is to provide forhybridization or PCR probes which are capable of detectingpolynucleotide sequences, including genomic sequences, encoding H-nm23or closely related molecules. The specificity of the probe, whether itis made from a highly specific region, e.g., 10 unique nucleotides inthe 5′ regulatory region, or a less specific region, e.g., especially inthe 3′ region, and the stringency of the hybridization or amplification(maximal, high, intermediate or low) will determine whether the probeidentifies only naturally occurring H-nm23, alleles or relatedsequences.

[0121] Probes may also be used for the detection of related sequencesand should preferably contain at least 50% of the nucleotides from anyof these H-nm23 encoding sequences. The hybridization probes of thesubject invention may be derived from the nucleotide sequence of SEQ IDNO:2 or from genonic sequence including promoter, enhancer elements andintrons of the naturally occurring H-nm23. Hybridization probes may belabeled by a variety of reporter groups, including radionuclides such as³²P or ³⁵S, or enzymatic labels such as alkaline phosphatase coupled tothe probe via avidin/biotin coupling systems, and the like.

[0122] Other means for producing specific hybridization probes forH-nm23 DNAs include the cloning of nucleic acid sequences encodingH-nm23 or H-nm23 derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art and are commercially availableand may be used to synthesize RNA probes in vitro by means of theaddition of the appropriate RNA polymerase as T7 or SP6 RNA polymeraseand the appropriate radioactively labeled nucleotides.

[0123] Diagnostic Use

[0124] Polynucleotide sequences encoding H-nm23 may be used for thediagnosis of conditions or diseases with which the expression of H-nm23is associated. For example, polynucleotide sequences encoding H-nm23 maybe used in hybridization or PCR assays of fluids or tissues frombiopsies to detect H-nm23 expression. The form of such qualitative orquantitative methods may include Southern or northern analysis, dot blotor other membrane-based technologies; PCR technologies; dip stick, pin,chip and ELISA technologies. All of these techniques are well known inthe art and are the basis of many commercially available diagnostickits.

[0125] The H-nm23 nucleotide sequence disclosed herein provide the basisfor assays that detect activation or induction associated with disease.The H-nm23 nucleotide sequence may be labeled by methods known in theart and added to a fluid or tissue sample from a patient underconditions suitable for the formation of hybridization complexes. Afteran incubation period, the sample is washed with a compatible fluid whichoptionally contains a dye (or other label requiring a developer) if thenucleotide has been labeled with an enzyme. After the compatible fluidis rinsed off, the dye is quantitated and compared with a standard. Ifthe amount of dye in the biopsied or extracted sample is significantlyelevated over that of a comparable control sample, the nucleotidesequence has hybridized with nucleotide sequences in the sample, and thepresence of elevated levels of H-nm23 nucleotide sequences in the sampleindicates the presence of the associated inflammation and/or disease.

[0126] Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regime in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient. Inorder to provide a basis for the diagnosis of disease, a normal orstandard profile for H-nm23 expression must be established. This isaccomplished by combining body fluids or cell extracts taken from normalsubjects, either animal or human, with H-nm23, or a portion thereof,under conditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fornormal subjects with a dilution series of H-nm23 run in the sameexperiment where a known amount of substantially purified H-nm23 isused. Standard values obtained from normal samples may be compared withvalues obtained from samples from patients afflicted withH-nm23-associated diseases. Deviation between standard and subjectvalues is used to establish the presence of disease.

[0127] Once disease is established, a therapeutic agent is administeredand a treatment profile is generated. Such assays may be repeated on aregular basis to evaluate whether the values in the profile progresstoward or return to the normal or standard pattern. Successive treatmentprofiles may be used to show the efficacy of treatment over a period ofseveral days or several months.

[0128] Polymerase Chain Reaction (PCR) as described in U.S. Pat. Nos.4,683,195 and 4,965,188 provides additional uses for oligonucleotidesbased upon the H-nm23 sequence. Such oligomers are generally chemicallysynthesized, but they may be generated enzymatically or produced from arecombinant source. Oligomers generally comprise two nucleotidesequences, one with sense orientation (5′->3′) and one with antisense(3′<-5′), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

[0129] Additionally, methods which may be used to quantitate theexpression of a particular molecule include radiolabeling (Melby P C etal 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al1993 Anal Biochem 229-36) nucleotides, coamplification of a controlnucleic acid, and standard curves onto which the experimental resultsare interpolated. Quantitation of multiple samples may be speeded up byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation. A definitive diagnosis of this typemay allow health professionals to begin aggressive treatment and preventfurther worsening of the condition. Similarly, further assays can beused to monitor the progress of a patient during treatment. Furthermore,the nucleotide sequences disclosed herein may be used in molecularbiology techniques that have not yet been developed, provided the newtechniques rely on properties of nucleotide sequences that are currentlyknown such as the triplet genetic code, specific base pair interactions,and the like.

[0130] Therapeutic Use

[0131] Based upon its homology to the genes encoding the nm23 isoformsand its expression profile, the H-nm23 polynucleotide disclosed hereinmay provide the basis for the design of molecules for the treatment ofcancer or other disorders associated with abnormal tissue development ordifferentiation.

[0132] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmid, may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisenseH-nm23. See, for example, the techniques described in Sambrook et al(supra) and Ausubel et al (supra).

[0133] The polynucleotides comprising full length cDNA sequence and/orits regulatory elements enable researchers to use H-nm23 as aninvestigative tool in sense (Youssoufian H and H F Lodish 1993 Mol CellBiol 13:98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem60:631-652) regulation of gene function. Such technology is now wellknown in the art, and sense or antisense oligomers, or larger fragments,can be designed from various locations along the coding or controlregions.

[0134] Genes encoding H-nm23 can be turned off by transfecting a cell ortissue with expression vectors which express high levels of a desiredH-nm23 fragment. Such constructs can flood cells with untranslatablesense or antisense sequences. Even in the absence of integration intothe DNA, such vectors may continue to transcribe RNA molecules until allcopies are disabled by endogenous nucleases. Transient expression maylast for a month or more with a non-replicating vector (Mettler I,personal communication) and even longer if appropriate replicationelements are part of the vector system.

[0135] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA or PNA, to thecontrol regions of H-nm23, i.e., the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, e.g.,between −10 and +10 regions of the leader sequence, are preferred. Theantisense molecules may also be designed to block translation of mRNA bypreventing the transcript from binding to ribosomes. Similarly,inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing compromises the ability of the doublehelix to open sufficiently for the binding of polymerases, transcriptionfactors, or regulatory molecules. Recent therapeutic advances usingtriplex DNA were reviewed by Gee J E et al (In: Huber B E and B I Carr(1994) Molecular and Immunologic Approaches, Futura Publishing Co, MtKisco, N.Y.).

[0136] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Withinthe scope of the invention are engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of RNA encoding H-nm23.

[0137] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0138] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of RNAmolecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding H-nm23. Such DNA sequences maybe incorporated into a wide variety of vectors with suitable RNApolymerase promoters such as T7 or SP6. Alternatively, antisense cDNAconstructs that synthesize antisense RNA constitutively or inducibly canbe introduced into cell lines, cells or tissues.

[0139] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine and wybutosine as well as acetyl-, methyl-, thio- andsimilarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0140] Methods for introducing vectors into cells or tissues includethose methods discussed infra and which are equally suitable for invivo, in vitro and ex vivo therapy. For ex vivo therapy, vectors areintroduced into stem cells taken from the patient and clonallypropagated for autologous transplant back into that same patient ispresented in U.S. Pat. Nos. 5,399,493 and 5,437,994, disclosed herein byreference. Delivery by transfection and by liposome are quite well knownin the art.

[0141] Furthermore, the nucleotide sequences for H-nm23 disclosed hereinmay be used in molecular biology techniques that have not yet beendeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including but not limited to suchproperties as the triplet genetic code and specific base pairinteractions.

[0142] Detection and Mapping of Related Polynucleotide Sequences

[0143] The nucleic acid sequence for H-nm23 can also be used to generatehybridization probes for mapping the naturally occurring genomicsequence. The sequence may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Theseinclude in situ hybridization to chromosomal spreads, flow-sortedchromosomal preparations, or artificial chromosome constructions such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price C M (1993; Blood Rev 7:127-34) and Trask B J (1991;Trends Genet 7:149-54).

[0144] The technique of fluorescent in situ hybridization of chromosomespreads has been described, among other places, in Verma et al (1988)Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, NewYork N.Y. Fluorescent in situ hybridization of chromosomal preparationsand other physical chromosome mapping techniques may be correlated withadditional genetic map data. Examples of genetic map data can be foundin the 1994 Genome Issue of Science (265:1981 f). Correlation betweenthe location of a H-nm23 on a physical chromosomal map and a specificdisease (or predisposition to a specific disease) may help delimit theregion of DNA associated with that genetic disease. The nucleotidesequences of the subject invention may be used to detect differences ingene sequences between normal, carrier or affected individuals.

[0145] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. For example,an STS based map of the human genome was recently published by thewhitehead-MIT Center for Genomic Research (Hudson T J et al (1995)Science 270:1945-1954). Often the placement of a gene on the chromosomeof another mammalian species such as mouse (Whitehead Institute/MITCenter for Genome Research, Genetic Map of the Mouse, Database Release10, Apr. 28, 1995) may reveal associated markers even if the number orarm of a particular human chromosome is not known. New sequences can beassigned to chromosomal arms, or parts thereof, by physical mapping.This provides valuable information to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once a disease or syndrome, such as ataxia telangiectasia(AT), has been crudely localized by genetic linkage to a particulargenomic region, for example, AT to 11q22-23 (Gatti et al (1988) Nature336:577-580), any sequences mapping to that area may representassociated or regulatory genes for further investigation. The nucleotidesequence of the subject invention may also be used to detect differencesin the chromosomal location due to translocation, inversion, etc. amongnormal, carrier or affected individuals.

[0146] Pharmaceutical Compostions

[0147] The present invention relates to pharmaceutical compositionswhich may comprise nucleotides, proteins, antibodies, agonists,antagonists, or inhibitors, alone or in combination with at least oneother agent, such as stabilizing compound, which may be administered inany sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Any of thesemolecules can be administered to a patient alone, or in combination withother agents, drugs or hormones, in pharmaceutical compositions where itis mixed with excipient(s) or pharmaceutically acceptable carriers. Inone embodiment of the present invention, the pharmaceutically acceptablecarrier is pharmaceutically inert.

[0148] Administration of Pharmaceutical Compositions

[0149] Administration of pharmaceutical compositions is accomplishedorally or parenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tumor), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition to the activeingredients, these pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Maack Publishing Co,Easton, Pa.).

[0150] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for ingestion by the patient.

[0151] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0152] Dragee cores are provided with suitable coatings such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

[0153] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

[0154] Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active compoundsmay be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

[0155] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0156] Manufacture and Storage

[0157] The pharmaceutical compositions of the present invention may bemanufactured in a manner that known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

[0158] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

[0159] After pharmaceutical compositions comprising a compound of theinvention formulated in a acceptable carrier have been prepared, theycan be placed in an appropriate container and labeled for treatment ofan indicated condition. For administration of H-nm23, such labelingwould include amount, frequency and method of administration.

[0160] Therapeutically Effective Dose

[0161] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve the intended purpose. Thedetermination of an effective dose is well within the capability ofthose skilled in the art.

[0162] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model is also used to achieve a desirable concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

[0163] A therapeutically effective dose refers to that amount of proteinor its antibodies, antagonists, or inhibitors which ameliorate thesymptoms or condition. Therapeutic efficacy and toxicity of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., ED50 (the dosetherapeutically effective in 50% of the population) and LD50 (the doselethal to 50% of the population). The dose ratio of toxic to therapeuticeffects is the therapeutic index, which can be expressed as theLD₅₀/ED₅₀ ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use. The dosage of such compounds lies preferably within a rangeof circulating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0164] The exact dosage is chosen by the individual physician in view ofthe patient to be treated. Dosage and administration are adjusted toprovide sufficient levels of the active moiety or to maintain thedesired effect. Additional factors which may be taken into accountinclude the severity of the disease state, e.g., tumor size andlocation; age, weight and gender of the patient; diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long actingpharmaceutical compositions might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular formulation.

[0165] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature. See U.S. Pat. Nos. 4,657,760;5,206,344; or 5,225,212. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0166] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0167] I cDNA Library Construction

[0168] The normal breast (BRSTNOT05) and breast carcinoma (BRSTTUT03)cDNA libraries were constructed from the breast tissue of a 58 year oldCaucasian female. The patient was diagnosed with multicentric invasivegrade 4 lobular carcinoma. The normal breast tissue is adjacent,microscopically normal tissue.

[0169] The frozen tissue was homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.0,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water and DNase treated at 37° C. The RNAextraction was repeated with acid phenol chloroform pH 8.0 andprecipitated with sodium acetate and ethanol as before. The mRNA wasthen isolated using the Qiagen Oligotex kit (QIAGEN Inc; Chatsworth,Calif.) and used to construct the cDNA library.

[0170] The mRNA was handled according to the recommended protocols inthe SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(Cat. #18248-013; Gibco/BRL), cDNAs were fractionated on a SepharoseCL4B column (Cat. #275105-01; Pharmacia), and those cDNAs exceeding 400bp were ligated into the PSPORT1 plasmid. The plasmid PSPORT1 wassubsequently transformed into DHSα competent cells (Cat. #18258-012;Gibco/BRL).

[0171] II Isolation and Sequencing of cDNA Clones

[0172] Plasmid DNA was released from the cells and purified using theREAL Prep 96 Plasmid Kit for Rapid Extraction Alkaline Lysis PlasmidMinipreps (Catalog #26173; QIAGEN, Inc). This kit enables thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Catalog #22711, LIFE TECHNOLOGIES™) withcarbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, thecultures were incubated for 19 hours and at the end of incubation, thecells were lysed with 0.3 ml of lysis buffer; and 3) followingisopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1ml of distilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

[0173] The cDNAs were sequenced by the method of Sanger F and A RCoulson (1975; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200(Hamilton, Reno, Nev.) in combination with Peltier Thermal Cyclers(PTC200 from M J Research, Watertown, Mass.) and Applied Biosystems 377DNA Sequencing Systems; and the reading frame was determined.

[0174] III Homology Searching of cDNA Clones and their Deduced Proteins

[0175] Each cDNA was compared to sequences in GenBank using a searchalgorithm developed by Applied Biosystems and incorporated into theINHERIT 670 sequence analysis system. In this algorithm, PatternSpecification Language (TRW Inc, Los Angeles, Calif.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

[0176] Peptide and protein sequence homologies were ascertained usingthe INEHERIT 670 sequence analysis system in a way similar to that usedin DNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

[0177] BLAST, which stands for Basic Local Alignment Search Tool(Altschul S F (1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) JMol Biol 215:403-10), was used to search for local sequence alignments.BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs. BLAST is useful for matches which do notcontain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

[0178] An HSP consists of two sequence fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

[0179] IV Northern Analysis

[0180] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al. supra).

[0181] Analogous computer techniques use BLAST (Altschul S F 1993 and1990, supra) to search for identical or related molecules in nucleotidedatabases such as GenBank or the LIFESEQ™ database (Incyte, Palo Alto,Calif.). This analysis is much faster than multiple, membrane-basedhybridizations. In addition, the sensitivity of the computer search canbe modified to determine whether any particular match is categorized asexact or homologous.

[0182] The basis of the search is the product score which is defined as:

% sequence identity×% maximum BLAST score/100

[0183] and it takes into account both the degree of similarity betweentwo sequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; and at70, the match will be exact. Homologous molecules are usually identifiedby selecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules.

[0184] The results of the search are reported as a list of libraries inwhich the full length sequence, or parts thereof, is represented, theabundance of the sequence, and the percent abundance. Abundance directlyreflects the number of times a particular transcript is present in acDNA library, and percent abundance is abundance divided by the totalnumber of sequences examined in the library.

[0185] V Extension of H-nm23 to Full Length or to Recover RegulatoryElements

[0186] The nucleic acid sequence encoding full length H-nm23 (SEQ IDNO:2) is used to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ sequences fromgenomic libraries. One primer is synthesized to initiate extension inthe antisense direction (XLR) and the other is synthesized to extendsequence in the sense direction (XLF). Primers allow the extension ofthe known H-nm23 nucleotide sequence “outward” generating ampliconscontaining new, unknown nucleotide sequence for the region of interest(U.S. patent application Ser. No. 08/487,112, filed Jun. 7, 1995,specifically incorporated by reference). The initial primers aredesigned from the cDNA using OLIGO® 4.06 Primer Analysis Software(National Biosciences), or another appropriate program, to be 22-30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68 °-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations is avoided.

[0187] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0188] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M J Research,Watertown, Mass.) and the following parameters:

[0189] Step 1 94° C. for 1 min (initial denaturation)

[0190] Step 2 65° C. for min

[0191] Step 3 68° C. for 6 min

[0192] Step 4 94° C. for 15 sec

[0193] Step 5 65° C. for 1 min

[0194] Step 6 68° C. for 7 min

[0195] Step 7 Repeat step 4-6 for 15 additional cycles

[0196] Step 8 94° C. for 15 sec

[0197] Step 9 65° C. for 1 min

[0198] Step 10 68° C. for 7:15 min

[0199] Step 11 Repeat step 8-10 for 12 cycles

[0200] Step 12 72° C. for 8 min

[0201] Step 13 4° C. (and holding)

[0202] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were selected and cut outof the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK (QIAGEN Inc). After recovery of theDNA, Klenow enzyme was used to trim single-stranded, nucleotideoverhangs creating blunt ends which facilitate religation and cloning.

[0203] After ethanol precipitation, the products are redissolved in 13,μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1μT4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook J et al, supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook J et al, supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

[0204] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions:

[0205] Step 1 94° C. for 60 sec

[0206] Step 2 94° C. for 20 sec

[0207] Step 3 55° C. for 30 sec

[0208] Step 4 72° C. for 90 sec

[0209] Step 5 Repeat steps 2-4 for an additional 29 cycles

[0210] Step 6 72° C. for 180 sec

[0211] Step 7 4° C. (and holding)

[0212] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid and sequenced.

[0213] VI Labeling and Use of Hybridization Probes

[0214] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 mCi of [γ-³²P] adenosine triphosphate(Amersham, Chicago, Ill.) and T4 polynucleotide kinase (DuPont NEN®,Boston, Mass.). The labeled oligonucleotides are substantially purifiedwith Sephadex G-25 super fine resin column (Pharmacia). A portioncontaining 10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPontNEN®).

[0215] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots or the blots areexposed in a Phosphorlmager cassette (Molecular Dynamics, Sunnyvale,Calif.) for several hours, hybridization patterns are compared visually.

[0216] VII Antisense Molecules

[0217] The nucleotide sequence encoding H-nm23, or any part thereof, isused to inhibit in vivo or in vitro expression of naturally occurringH-nm23. Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on the codingsequence of H-nm23 as shown in FIGS. 1A, 1B, and 1C is used to inhibitexpression of naturally occurring H-nm23. The complementaryoligonucleotide is designed from the most unique 5′ sequence as shown inFIGS. 1A, 1B, and 1C and used either to inhibit transcription bypreventing promoter binding to the upstream nontranslated sequence ortranslation of an H-nm23 transcript by preventing the ribosome frombinding. Using an appropriate portion of the leader and 5′ sequence ofSEQ ID NO:2, an effective antisense oligonucleotide includes any 15-20nucleotides spanning the region which translates into the signal orearly coding sequence of the polypeptide as shown in FIGS. 1A, 1B, and1C.

[0218] VIII Expression of H-nm23

[0219] Expression of H-nm23 is accomplished by subcloning the cDNAs intoappropriate vectors and transfecting the vectors into host cells. Inthis case, the cloning vector, PSPORT1, previously used for thegeneration of the cDNA library is used to express H-nm23 in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Metand the subsequent 7 residues of β-galactosidase. Immediately followingthese eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0220] Induction of an isolated, transfected bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst seven residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length H-nm23. The signal sequence directs thesecretion of H-nm23 into the bacterial growth media which can be useddirectly in the following assay for activity.

[0221] IX H-nm23 Activity

[0222] Binding of H-nm23 to DNA is assayed by monitoring the differencesin electrophoretic mobility of the DNA with and without protein bound. ADNA probe (for example, a 105 base pair double-stranded fragmentcomprising the c-myc promoter region) is radiolabeled with ³²P by thetransfer of the terminal phosphate of [g-³²P]ATP to the 5′ ends of theDNA, catalyzed by T4 polynucleotide kinase (New England Biolabs,Beverly, Mass.). The radiolabeled DNA is incubated with varyingconcentrations of H-nm23 at room temperature for approximately 1 hour.The incubation reactions are electrophoresed through non-denaturingpolyacrylamide gels. The gels are dried and autoradiographed. Therelative intensities of the bands corresponding to uncomplexed DNA andprotein-complexed DNA for each concentration of added H-nm23 aredetermined by densitometry. The resulting titration curve is used alongwith the concentrations of the radiolabeled DNA and added H-nm23 tocalculate values for the affinity of H-nm23 for the DNA.

[0223] The NDPK activity of H-nm23 is measured in a coupled pyruvatekinase-lactate dehydrogenase assay system (Sigma Chemical Co., St.Louis, Mo.), with ATP as the phosphate donor and dTDP as the phosphateacceptor nucleotides. The ADP produced in the NDPK reaction reacts withphosphoenolpyruvate via the pyruvate kinase to form pyruvate. In thepresence of pyruvate, the lactate dehydrogenase oxidizes NADH. NADHoxidation is accompanied by a change in absorbance at 340 nm, which ismonitored in a spectrophotometer.

[0224] X Production of H-nm23 Specific Antibodies

[0225] H-nm23 substantially purified using PAGE electrophoresis(Sambrook, supra) is used to immunize rabbits and to produce antibodiesusing standard protocols. The amino acid sequence translated from H-nm23is analyzed using DNASTAR software (DNAStar Inc) to determine regions ofhigh immunogenicity and a corresponding oligopolypeptide is synthesizedand used to raise antibodies by means known to those of skill in theart. Analysis to select appropriate epitopes, such as those near theC-terminus or in hydrophilic regions (shown in FIG. 3) is described byAusubel F M et al (supra).

[0226] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing finoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccininide ester(MBS; Ausubel F M et al, supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

[0227] XI Purification of Naturally Occurring H-nm23 Using SpecificAntibodies

[0228] Naturally occurring or recombinant H-nm23 is substantiallypurified by immunoaffinity chromatography using antibodies specific forH-nm23. An immunoaffinity column is constructed by covalently couplingH-nm23 antibody to an activated chromatographic resin such asCNBr-activated Sepharose (Pharmacia Biotech). After the coupling, theresin is blocked and washed according to the manufacturer'sinstructions.

[0229] Cellular fractions from cells containing H-nm23 are prepared bysolubilization of the whole cell and isolation of subcellular fractionsby differential centrifugation, by the addition of detergent, or byother methods well known in the art. Alternatively, soluble H-nm23containing a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

[0230] A fractionated H-nm23-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of H-nm23 (e.g., high ionic strengthbuffers in the presence of detergent). The column is eluted underconditions that disrupt antibody/H-nm23 binding (e.g., a buffer of pH2-3 or a high concentration of a chaotrope such as urea or thiocyanateion), and H-nm23 is collected.

[0231] XII Identification of Molecules Which Interact with H-nm23

[0232] H-nm23 is useful as a research tools for identification,characterization and purification of molecules with which it interacts.In one embodiment of affinity purification, H-nm23 is covalently coupledto a chromatography column. Cells and their membranes are extracted,endogenous H-nm23 is removed and various H-nm23-free subcomponents arepassed over the column. H-nm23-associated molecules bind to the columnby virtue of their biological affinity. The H-nm23-complex is recoveredfrom the column, dissociated and the recovered molecule is subjected toN-terminal protein sequencing, nucleic acid sequencing, orhigh-performance liquid chromatography/mass spectrometry (HPLC/MS),depending on the type of molecule. The amino acid or nucleotide sequenceor mass spectral analysis is then used to identify the captured moleculeor, in the case of a protein ligand, to design degenerateoligonucleotide probes for cloning its gene from an appropriate cDNAlibrary.

[0233] In an alternate method, monoclonal antibodies are raised againstH-nm23 and screened to identify those compounds which inhibit thebinding of the antibody to H-nm23. These monoclonal antibodies may thenused in affinity purification or expression cloning of associatedmolecules.

[0234] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 5 187 amino acids amino acid single linear peptide <Unknown> Consensus1 Met Gly Gly Leu Phe Trp Arg Ser Ala Leu Arg Gly Leu Arg Cys Gly 1 5 1015 Pro Arg Ala Pro Gly Pro Ser Leu Leu Val Arg His Gly Ser Gly Gly 20 2530 Pro Ser Trp Thr Arg Glu Arg Thr Leu Val Ala Val Lys Pro Asp Gly 35 4045 Val Gln Arg Arg Leu Val Gly Asp Val Ile Gln Arg Phe Glu Arg Arg 50 5560 Gly Phe Thr Leu Val Gly Met Lys Met Leu Gln Ala Pro Glu Ser Val 65 7075 80 Leu Ala Glu His Tyr Gln Asp Leu Arg Arg Lys Pro Phe Tyr Pro Ala 8590 95 Leu Ile Arg Tyr Met Ser Ser Gly Pro Val Val Ala Met Val Trp Glu100 105 110 Gly Tyr Asn Val Val Arg Ala Ser Arg Ala Met Ile Gly His ThrAsp 115 120 125 Ser Ala Glu Ala Ala Pro Gly Thr Ile Arg Gly Asp Phe SerVal His 130 135 140 Ile Ser Arg Asn Val Ile His Ala Ser Asp Ser Val GluGly Ala Gln 145 150 155 160 Arg Glu Ile Gln Leu Trp Phe Gln Ser Ser GluLeu Val Ser Trp Ala 165 170 175 Asp Gly Gly Gln His Ser Ser Ile His ProAla 180 185 986 base pairs nucleic acid single linear cDNA <Unknown>Consensus 2 GGCCGGGCGT CATGGGCGGC CTCTTCTGGC GCTCCGCGCT GCGGGGGCTGCGCTGCGGCC 60 CGCGGGCCCC GGGCCCGAGC CTGCTAGTGC GCCACGGCTC GGGAGGGCCCTCCTGGACCC 120 GGGAGCGGAC CCTGGTGGCG GTGAAGCCCG ATGGCGTGCA ACGGCGGCTCGTTGGGGACG 180 TGATCCAGCG CTTTGAGAGG CGGGGCTTCA CGCTGGTGGG GATGAAGATGCTGCAAGCAC 240 CAGAGAGCGT CCTTGCCGAG CACTACCAGG ACCTGCGGAG GAAGCCCTTCTACCCTGCCC 300 TMATCCGCTA CATGAGCTCT GGGCCTGTGG TGGCCATGGT CTGGGAAGGGTACAATGTCG 360 TCCGCGCCTC RAGGGCCATG ATTGGACACA CCGACTCGGC TGAGGCTGCCCCAGGAACCA 420 TAAGGGGTGA CTTCAGCGTC CACATCAGCA GGAATGTCAT CCACGCCAGCGACTCCGTGG 480 AGGGGGCCCA GCGGGAGATC CAGCTGTGGT TCCAGAGCAG TGAGCTGGTGAGCTGGGCAG 540 ACGGGGGCCA GCACAGCAGC ATCCACCCAG CCTGAGGCTC AAGCTGCCCTTACCACCCCA 600 TCCCCCACGC AGGACCAACT ACCTCCGTCA GCAAGAACCC AAGCCCACATCCAAACCTGC 660 CTGTCCCAAA CCACTTACTT CCCTGTTCAC CTCTGCCCCA CCCCAGCCCAGAGGAGTTTG 720 AGCCACCAAC TTCAGTGCCT TTCTGTACCC CAAGCCAGCA CAAGATTGGACCAATCCTTT 780 TTGCACCAAA GTGCCGGACA ACCTTTGTGG TGGGGGGGGG TCTTCACATTATCATAACCT 840 CTCCTCTAAA GGGGAGGCAT TAAAATTCAC TGTGCCCAGC ACATGGGTGGTACACTAATT 900 ATGACTTCCC CCAGCTCTGA GGTAGAAATG ACGCCTTTAT GCAAGTTGTAAGGAGTTGAA 960 CAGTAAAGAG GAAGTTTTGC ACACCC 986 168 amino acids aminoacid single linear peptide GenBank 1051256 3 Met Ile Cys Leu Val Leu ThrIle Phe Ala Asn Leu Phe Pro Ala Ala 1 5 10 15 Cys Thr Gly Ala His GluArg Thr Phe Leu Ala Val Lys Pro Asp Gly 20 25 30 Val Gln Arg Arg Leu ValGly Glu Ile Val Arg Arg Phe Glu Arg Lys 35 40 45 Gly Phe Lys Leu Val AlaLeu Lys Leu Val Gln Ser Ser Glu Glu Leu 50 55 60 Leu Arg Glu His Tyr AlaGlu Leu Arg Glu Arg Pro Phe Tyr Gly Arg 65 70 75 80 Leu Val Lys Tyr MetAla Ser Gly Pro Val Val Ala Met Val Trp Gln 85 90 95 Gly Leu Asp Val ValArg Thr Ser Arg Ala Leu Ile Gly Ala Thr Asn 100 105 110 Pro Ala Asp AlaPro Pro Gly Thr Ile Arg Gly Asp Phe Cys Ile Glu 115 120 125 Val Gly AsnLeu Ile His Gly Ser Asp Ser Val Glu Ser Ala Arg Arg 130 135 140 Glu IleAla Leu Trp Phe Arg Ala Asp Glu Leu Leu Cys Trp Glu Asp 145 150 155 160Ser Ala Gly His Trp Leu Tyr Glu 165 152 amino acids amino acid singlelinear peptide GenBank 468542 4 Met Ala Asn Cys Glu Arg Thr Phe Ile AlaIle Lys Pro Asp Gly Val 1 5 10 15 Gln Arg Gly Leu Val Gly Glu Ile IleLys Arg Phe Glu Gln Lys Gly 20 25 30 Phe Arg Leu Val Gly Leu Lys Phe MetGln Ala Ser Glu Asp Leu Leu 35 40 45 Lys Glu His Tyr Val Asp Leu Lys AspArg Pro Phe Phe Ala Gly Leu 50 55 60 Val Lys Tyr Met His Ser Gly Pro ValVal Ala Met Val Trp Glu Gly 65 70 75 80 Leu Asn Val Val Lys Thr Gly ArgVal Met Leu Gly Glu Thr Asn Pro 85 90 95 Ala Asp Ser Lys Pro Gly Thr IleArg Gly Asp Phe Cys Ile Gln Val 100 105 110 Gly Arg Asn Ile Ile His GlySer Asp Ser Val Glu Ser Ala Glu Lys 115 120 125 Glu Ile Gly Leu Trp PheHis Pro Glu Glu Leu Val Asp Tyr Thr Ser 130 135 140 Cys Ala Gln Asn TrpIle Tyr Glu 145 150 152 amino acids amino acid single linear peptideGenBank 127983 5 Met Ala Asn Leu Glu Arg Thr Phe Ile Ala Ile Lys Pro AspGly Val 1 5 10 15 Gln Arg Gly Leu Val Gly Glu Ile Ile Lys Arg Phe GluGln Lys Gly 20 25 30 Phe Arg Leu Val Ala Met Lys Phe Leu Arg Ala Ser GluGlu His Leu 35 40 45 Lys Gln His Tyr Ile Asp Leu Lys Asp Arg Pro Phe PhePro Gly Leu 50 55 60 Val Lys Tyr Met Asn Ser Gly Pro Val Val Ala Met ValTrp Glu Gly 65 70 75 80 Leu Asn Val Val Lys Thr Gly Arg Val Met Leu GlyGlu Thr Asn Pro 85 90 95 Ala Asp Ser Lys Pro Gly Thr Ile Arg Gly Asp PheCys Ile Gln Val 100 105 110 Gly Arg Asn Ile Ile His Gly Ser Asp Ser ValLys Ser Ala Glu Lys 115 120 125 Glu Ile Ser Leu Trp Phe Lys Pro Glu GluLeu Val Asp Tyr Lys Ser 130 135 140 Cys Ala His Asp Trp Val Tyr Glu 145150

1. An isolated polypeptide selected from the group consisting of: a) apolypeptide comprising an amino acid sequence of SEQ ID NO:1, b) apolypeptide comprising a naturally occurring an amino acid sequence atleast 90% identical to an amino acid sequence of SEQ ID NO:1, c) abiologically active fragment of a polypeptide having an amino acidsequence of SEQ ID NO:1, and d) an immunogenic fragment of a polypeptidehaving an amino acid sequence of SEQ ID NO:1.
 2. An isolated polypeptideof claim 1, comprising an amino acid sequence of SEQ ID NO:1:
 3. Anisolated polynucleotide encoding a polypeptide of claim
 1. 4. Anisolated polynucleotide encoding a polypeptide of claim
 2. 5. Anisolated polynucleotide of claim 4, having a sequence of SEQ ID NO:2. 6.A recombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide of claim
 3. 7. A cell transformed with arecombinant polynucleotide of claim
 6. 8. A transgenic organismcomprising a recombinant polynucleotide of claim
 6. 9. A method ofproducing a polypeptide of claim 1, the method comprising: a) culturinga cell under conditions suitable for expression of the polypeptide,wherein said cell is transformed with a recombinant polynucleotide, andsaid recombinant polynucleotide comprises a promoter sequence operablylinked to a polynucleotide encoding the polypeptide of claim 1, and b)recovering the polypeptide so expressed.
 10. A method of claim 9,wherein the polypeptide comprises the amino acid sequence of SEQ IDNO:1.
 11. An isolated antibody which specifically binds to a polypeptideof claim
 1. 12. An isolated polynucleotide selected from the groupconsisting of: a) a polynucleotide comprising a polynucleotide sequenceof SEQ ID NO:2, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical to a polynucleotidesequence of SEQ ID NO:2, c) a polynucleotide complementary to apolynucleotide of a), d) a polynucleotide complementary to apolynucleotide of b) and e) an RNA equivalent of a)-d).
 13. An isolatedpolynucleotide comprising at least 60 contiguous nucleotides of apolynucleotide of claim
 12. 14. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) hybridizingthe sample with a probe comprising at least 20 contiguous nucleotidescomprising a sequence complementary to said target polynucleotide in thesample, and which probe specifically hybridizes to said targetpolynucleotide, under conditions whereby a hybridization complex isformed between said probe and said target polynucleotide or fragmentsthereof, and b) detecting the presence or absence of said hybridizationcomplex, and, optionally, if present, the amount thereof.
 15. A methodof claim 14, wherein the probe comprises at least 60 contiguousnucleotides.
 16. A method of detecting a target polynucleotide in asample, said target polynucleotide having a sequence of a polynucleotideof claim 12, the method comprising: a) amplifying said targetpolynucleotide or fragment thereof using polymerase chain reactionamplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises an amino acidsequence of SEQ ID NO:1.
 19. A method for treating a disease orcondition associated with decreased expression of functional H-nm23,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional H-nm23, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method of screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional H-nm23, comprising administering to apatient in need of such treatment a composition of claim
 24. 26. Amethod of screening for a compound that specifically binds to thepolypeptide of claim 1, the method comprising: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 27. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, saidmethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of SEQ ID NO:2, themethod comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 29. A method of assessing toxicityof a test compound, the method comprising: a) treating a biologicalsample containing nucleic acids with the test compound, b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of H-nm23 in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of H-nm23 in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofH-nm23 in a subject, comprising administering to said subject aneffective amount of the composition of claim
 34. 36. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of an amino acid sequence of SEQ ID NO:1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibodies from said animal, and c) screening theisolated antibodies with the polypeptide, thereby identifying apolyclonal antibody which binds specifically to a polypeptide comprisingan amino acid sequence of SEQ ID NO:1.
 37. A polyclonal antibodyproduced by a method of claim
 36. 38. A composition comprising thepolyclonal antibody of claim 37 and a suitable carrier.
 39. A method ofmaking a monoclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of an amino acid sequence of SEQ ID NO:1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibody producing cells from the animal, c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells, d) culturing thehybridoma cells, and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide comprising an amino acidsequence of SEQ ID NO:1.
 40. A monoclonal antibody produced by a methodof claim
 39. 41. A composition comprising the monoclonal antibody ofclaim 40 and a suitable carrier.
 42. The antibody of claim 11, whereinthe monoclonal antibody is produced by screening a Fab expressionlibrary.
 43. The antibody of claim 11, wherein the antibody is producedby screening a recombinant immunoglobulin library.
 44. A method ofdetecting a polypeptide comprising an amino acid sequence of SEQ ID NO:1in a sample, the method comprising: a) incubating the antibody of claim11 with a sample under conditions to allow specific binding of theantibody and the polypeptide, and b) detecting specific binding, whereinspecific binding indicates the presence of a polypeptide comprising anamino acid sequence of SEQ ID NO:1 in the sample.
 45. A method ofpurifying a polypeptide comprising an amino acid sequence of SEQ ID NO:1from a sample, the method comprising: a) incubating the antibody ofclaim 11 with a sample under conditions to allow specific binding of theantibody and the polypeptide, and b) separating the antibody from thesample and obtaining the purified polypeptide comprising an amino acidsequence of SEQ ID NO:1.
 46. A microarray wherein at least one elementof the microarray is a polynucleotide of claim
 13. 47. A method ofgenerating an expression profile of a sample which containspolynucleotides, the method comprising: a) labeling the polynucleotidesof the sample, b) contacting the elements of the microarray of claim 46with the labeled polynucleotides of the sample under conditions suitablefor the formation of a hybridization complex, and c) quantifying theexpression of the polynucleotides in the sample.
 48. An array comprisingdifferent nucleotide molecules affixed in distinct physical locations ona solid substrate, wherein at least one of said nucleotide moleculescomprises a first oligonucleotide or polynucleotide sequencespecifically hybridizable with at least 30 contiguous nucleotides of atarget polynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.